【摘要】：黑骨藤為蘿摩科(Asclepiasaceae)杠柳屬(Periploca)植物黑龍骨(Periploca forrestii Schltr.)的根及根莖,大部分生長于陰暗潮濕之處,或者有水源的峭壁之上;主要分布于我國西部及南部地區(qū)如貴州、廣西、四川、青海、西藏等地。黑骨藤全株入藥,具有通經(jīng)、活血、解毒、祛風(fēng)的功效,民間廣泛應(yīng)用于治療閉合性軟組織損傷、風(fēng)濕與類風(fēng)濕等疾病。中藥有效物質(zhì)的辨識(shí)一直是中藥現(xiàn)代化研究中的重點(diǎn)和難點(diǎn)。為了尋求一種黑骨藤藥效物質(zhì)辨識(shí)的方法,本論文在國家自然基金的資助下,于課題組前期對(duì)黑骨藤80%乙醇提取部位指紋圖譜及譜效關(guān)系探索性研究的基礎(chǔ)上,開展了相關(guān)藥效物質(zhì)辨識(shí)研究:通過對(duì)分離材料的篩選,優(yōu)選出大孔樹脂為分離材料,對(duì)黑骨藤80%乙醇提取部位組分群進(jìn)行分離制備;并對(duì)分離得到的各個(gè)組分群進(jìn)行體外抗過敏、體外抗氧化、乙酰膽堿酯酶抑制活性、細(xì)胞毒性等藥效學(xué)優(yōu)選研究,從而選出活性較強(qiáng)的組分;然后采用紫外吸收光譜和高效液相色譜法對(duì)其進(jìn)行藥效物質(zhì)辨識(shí)研究,結(jié)果顯示,組分中主要成分為黃酮類化合物;基于藥效物質(zhì)辨識(shí)的結(jié)果,以黑骨藤總黃酮提取率為指標(biāo)進(jìn)行超聲預(yù)處理微波提取工藝研究,優(yōu)化提取工藝條件,并進(jìn)一步對(duì)其進(jìn)行體外抗過敏活性驗(yàn)證。通過上述研究,為黑骨藤及其制劑的深度開發(fā)利用和質(zhì)量控制提供了科學(xué)依據(jù)和理論基礎(chǔ)。主要結(jié)果如下:1.黑骨藤有效成分群的分離與富集此部分研究對(duì)黑骨藤80%乙醇提取液進(jìn)行了分離制備:通過預(yù)實(shí)驗(yàn)得到大孔樹脂為最佳分離材料,并對(duì)溶劑洗脫系統(tǒng)進(jìn)行了優(yōu)化:采用5%、10%、15%、20%、25%、30%、35%至95%乙醇分段洗脫,通過高效液相色譜法檢測(cè)洗脫液出峰情況,對(duì)其進(jìn)行調(diào)整,合并收集相同組分,歷時(shí)6個(gè)月,分離富集了6段黑骨藤組分群A(18 g)、B(8 g)、C(5 g)、D(10 g)、E(10 g)、F(10 g)。根據(jù)組分群的高效液相色譜圖,與黑骨藤80%乙醇提取部位指紋圖譜進(jìn)行歸屬分析:指紋圖譜上標(biāo)記了8個(gè)特征峰,其歸屬關(guān)系為A(1、2)B(3)C(4、5)D(6)E(7、8)F(30-35 min成分群)。這為下一步組分群的藥效研究奠定了基礎(chǔ)。2.黑骨藤各組分群的藥效學(xué)優(yōu)選研究此部分研究中,以上述富集的黑骨藤80%乙醇提取部位組分群為基礎(chǔ),結(jié)合黑骨藤傳統(tǒng)藥效和現(xiàn)代化藥理活性研究進(jìn)展,選擇抗氧化活性(ABTS·+自由基清除能力、DPPH·自由基清除能力)、乙酰膽堿酯酶抑制活性、抗過敏活性、細(xì)胞毒性對(duì)其進(jìn)行藥效學(xué)優(yōu)選研究。結(jié)果顯示:ABTS·+自由基清除能力大小:EDBCAFG;DPPH·自由基清除能力大小:EFDCABG;乙酰膽堿酯酶抑制活性大小:EDFBACG;體外抗過敏活性大小為EACFBDG;對(duì)MH7A類風(fēng)濕關(guān)節(jié)炎細(xì)胞毒性大小為EABCDGF。課題組前期通過譜效關(guān)系研究推測(cè)的抗炎有效物質(zhì)峰為2、6、8號(hào),包含于A(1、2)、D(6)E(7、8)中,在此部分試驗(yàn)中,證實(shí)了2、8號(hào)峰位于的A、E段擁有較好的活性;在體外抗氧化與體外乙酰膽堿酯酶抑制活性中,E段的活性都為最強(qiáng),因此,E段組分群可作為后續(xù)藥效物質(zhì)辨識(shí)研究的對(duì)象。3.黑骨藤E段組分群藥效物質(zhì)辨識(shí)研究此部分研究以活性最優(yōu)的E段組分群為對(duì)象,采用紫外吸收光譜法和高效液相色譜法對(duì)其進(jìn)行藥效物質(zhì)辨識(shí)研究。E段高效液相色譜圖中主要有5個(gè)特征峰,通過樣品與對(duì)照品紫外吸收波長和高效液相色譜圖保留時(shí)間對(duì)比,指認(rèn)了兩個(gè)黃酮類化合物,分別是槲皮素-3-O-β-D-吡喃葡萄糖苷和槲皮素-3-O-α-L-吡喃阿拉伯糖苷。4.黑骨藤總黃酮提取工藝研究前述藥效學(xué)優(yōu)選出來活性最佳的E段組分群中含有黃酮類化合物,這為黑骨藤的進(jìn)一步開發(fā)利用提供了科學(xué)參考。本項(xiàng)研究中,采用超聲預(yù)處理微波提取技術(shù),通過單因素實(shí)驗(yàn)篩選出超聲預(yù)處理最佳時(shí)間25 min,運(yùn)用中心組合(BBD)響應(yīng)面法,以黑骨藤總黃酮提取率為指標(biāo)優(yōu)化提取工藝參數(shù):微波功率(W)、乙醇濃度(%)、液料比(m L·g-1)及微波時(shí)間(s),通過實(shí)驗(yàn)得到最佳提取工藝參數(shù)為:乙醇濃度59.92%、液料比21.24 m L·g-1、微波時(shí)間為209.53 s、微波功率274.14 W。在此工藝參數(shù)下,黑骨藤總黃酮提取率為9.11±0.08%,與微波提取法相比增加了14.30%,與超聲波提取法相比增加了19.86%;通過聚酰胺純化后,超聲預(yù)處理微波提取的黑骨藤總黃酮的抗過敏活性半抑制率為IC50=1.033 mg·m L-1,分別優(yōu)于超聲提取法(IC50=12.913 mg·m L-1)與微波提取法(IC50=1.762 mg·m L-1)。
[Abstract]:The roots and rhizomes of Periploca forrestii Schltr. grow mostly in shady and humid places or on cliffs with water sources. They are mainly distributed in Western and southern China, such as Guizhou, Guangxi, Sichuan, Qinghai and Tibet. In order to find a method for identifying the effective substances of the Chinese herbal medicine, this paper, with the support of the National Natural Fund of China, has been widely used in the treatment of closed soft tissue injury, rheumatism and rheumatoid diseases. On the basis of exploratory study on fingerprint and Spectrum-Effect relationship of 80% ethanol extract part of Celastrus vicina, the research group carried out identification of related pharmacodynamic substances. Through screening the separation materials, macroporous resin was selected as the separation material, and the fractions of 80% ethanol extract part of Celastrus vicina were separated and prepared. The constituents were selected by the pharmacodynamic optimization of anti-allergy, anti-oxidation, acetylcholinesterase inhibitory activity and cytotoxicity in vitro, and then identified by ultraviolet absorption spectroscopy and high performance liquid chromatography. According to the results of identification of the effective substances, the extraction rate of total flavonoids from Celastrus Vicia was used as the index to study the ultrasonic pretreatment microwave extraction process, optimize the extraction process conditions, and further verify its anti-allergic activity in vitro. The main results are as follows: 1. Separation and enrichment of the effective components of the vine were studied. The 80% ethanol extract of the vine was separated and prepared. The porous resin was obtained as the best separation material by preliminary experiment, and the solvent elution system was optimized: 5%, 10%, 15%, 20%, 25%, 30%, 35% to 95% ethanol was eluted by stages. The eluent peaking was detected by high performance liquid chromatography (HPLC), and the eluent was adjusted to collect the same components. After six months, the eluent components A (18 g), B (8 g), C (5 g), D (10 g), E (10 g), F (10 g) were separated and enriched. According to the high performance liquid chromatogram of the eluent group, the fingerprints of the eluent and 80% ethanol extract fraction of Celasted for six months. Eight characteristic peaks were labeled on the stripe map, and the attribution relationship was A(1,2)B(3)C(4,5)D(6)E(7,8)F(30-35 min constituent group). This laid a foundation for the further study on the pharmacodynamics of each constituent group. 2. The pharmacodynamics optimization of each constituent group was studied in this part. The antioxidant activity (ABTS (+) radical scavenging capacity, DPPH (+) radical scavenging capacity), acetylcholinesterase inhibitory activity, anti-allergic activity and cytotoxicity were selected to optimize the pharmacodynamics of Guteng. Scavenging capacity: EFDCABG; acetylcholinesterase inhibitory activity: EDFBACG; in vitro antiallergic activity: EACFBDG; cytotoxicity to MH7A rheumatoid arthritis: EABCDGF. The peak values of anti-inflammatory active substances predicted by the study group were 2,6,8 and included in A (1,2), D (6) E (7,8). The results showed that the A and E segments of the 2 and 8 peaks had good activity, and the E segment had the strongest activity in vitro and in vitro, so the E segment group could be used as the object of follow-up study on identification of effective substances. Two flavonoids, quercetin-3-O-beta, were identified by ultraviolet absorption spectroscopy and high performance liquid chromatography. Five characteristic peaks were identified in the E-segment high performance liquid chromatogram. Study on the extraction technology of total flavonoids from Celastrus nigra L. The flavonoids in E-segment group with the best pharmacodynamics were selected, which provided a scientific reference for the further development and utilization of Celastrus nigra L. In this study, ultrasonic pretreatment microwave extraction technology was used. The optimum time of ultrasonic pretreatment was 25 minutes by single factor experiment. The extraction parameters of total flavonoids were optimized by using central combination (BBD) response surface methodology with the extraction rate of total flavonoids as the index: microwave power (W), ethanol concentration (%), liquid-to-material ratio (m L.g-1) and microwave time (s). The optimum extraction parameters were obtained by experiment as follows: ethanol concentration 59. 92%, liquid-to-material ratio 21.24 m L g-1, microwave time 209.53 s, microwave power 274.14 W. Under these parameters, the total flavonoids extraction rate was 9.11 0.08%, 14.30% higher than that of microwave extraction method, and 19.86% higher than that of ultrasonic extraction method. The semi-inhibitory rate of allergic activity was IC50=1.033 mg/ml-1, which was superior to ultrasonic extraction (IC50=12.913 mg/ml-1) and microwave extraction (IC50=1.762 mg/ml-1).
【學(xué)位授予單位】：貴州師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657;R284.1

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